The entry of Ca2+ ions into the cytosol from the extracellular fluid and from endoplasmic reticulum (ER) stores is used as a signaling mechanism by virtually all cell types to regulate functions as diverse as electrical excitability, secretion, proliferation and cell death. Improved optical technology now enables visualization of a hierarchy of Ca2+ signaling events, ranging from openings of single-channel Ca2+-permeable channels ('fundamental'events), concerted openings of clustered channels ('elementary'events) and propagating Ca2+ waves. The localized free [Ca2+] elevations arising through individual and clustered channels serve autonomous signaling functions, and their activity may further be coordinated through Ca2+ diffusion and Ca2+-induced Ca2+ release to propagate global cellular Ca2+ waves. Fundamental and elementary events thus form hierarchical building blocks underlying the complex spatiotemporal Ca2+ signals that permit graded and selective regulation of cell functions. Elucidation of their generation, interaction and functional consequences is, therefore, pivotal to understand the physiological functioning of the ubiquitous Ca2+ messenger pathway and its involvement in disease. Our overall goal is to elucidate, at the single-channel level, how cells generate the hierarchy of Ca2+ signals and how disruptions in Ca2+ signaling may be involved in disease pathogenesis. We focus on physiological Ca2+ signals generated by the ubiquitous inositol trisphosphate second messenger pathway, and on the pathogenic Ca2+-permeable pores formed by amyloid oligomers implicated in Alzheimer's disease. By utilizing novel biophotonic tools that now enable the optical imaging of calcium flux through individual channels and the localization and tracking of channel proteins with nanometer precision we aim to: (i) Further refine optical and analytical techniques for simultaneously monitoring Ca2+ flux through hundreds of individual channels in the plasma membrane and ER of intact cells. (ii) Elucidate how the activity of individual IP3 receptors (IP3R) at a release site is orchestrated to generate elementary Ca2+ puffs. (iii) Employ superresolution imaging techniques to determine the nanoscopic spatial distribution of IP3R, and how this impacts their functioning. (iv) Simultaneously monitor Ca2+ flux and peptide stoichiometry of individual amyloid pores to study fundamental mechanisms of membrane incorporation, channel gating and ion permeation. PUBLIC HEALTH RELEVANCE: Calcium serves a 'life or death'function in virtually all cells of the body, regulating processes as diverse as the heartbeat and synaptic transmission between brain cells, and disorders in calcium signals are implicated in Alzheimer's and many other diseases. Our goal is to take advantage of recent advances in microscopy that now enable imaging of individual molecules in intact cells to elucidate the hierarchical mechanisms by which calcium signals are generated at levels from single channels to the whole cell, with the dual aims of better understanding their normal functioning and how disruptions in Ca2+ signaling may lead to disease.